The invention is directed to novel C2-symmetrical and unsymmetrical chemical libraries for use in protein and receptor homodimerization and heterodimerization studies by solution phase methods. The invention further relates to novel combinatorial methods for synthesizing such libraries of compounds.
Ligand-induced receptor and protein dimerization or oligomerization has emerged as a general mechanism for signal transduction. Members of several receptor families of significance for drug discovery have been established to utilize this mode of receptor activation. These include protein tyrosine kinase receptors (homo- or heterodimerization), cytokine receptors (homo- or heterodimerization), serine/threonine kinase receptors (hetero-oligomerization) and members of the TNF-receptor family (trimerization). Within the cytokine receptor superfamily, the best studied examples are the human growth hormone (hGHr), prolactin (PRLr) and erythropoietin (EPOr) receptors, which form homodimers upon binding with their endogenous ligands. Similarly, intracellular signal transduction often proceeds by ligand-induced protein-protein homo- or heterodimerization.
The fact that the certain receptors and proteins appear to bind their ligands utilizing small clusters of residues for the majority of the binding interaction has led to the expectation that small molecules may be capable of triggering a receptor response. It has been anticipated that the generation of detailed knowledge concerning the dimerization modes and ligand binding domains of single transmembrane domain receptors will provide a basis for the design of functional agonists as well as ligand antagonists. However, the noncontiguous and multiple binding domains involved in both the protein-protein and ligand-protein interactions make it difficult to assess the dimerization mode or ligand binding domains in the absence of three-dimensional structural information. This is especially true considering the size of the typical endogenous ligands including proteins such as EPO (166 residues) which themselves contain noncontiguous binding domains which interact with both subunits of the dimerized receptor. Consequently, the search for non-protein ligands has been addressed through the use of random screening procedures.
Recently, the successful identification of cyclic polypeptides with the capacity to mimic the action of EPO was reported, together with details of the intricate receptor-ligand and receptor-receptor interactions in the bound complex (Wrighton et al. Science 1996, 273, 458; Livnah et al. Science 1996, 273, 464) Although these results represent a major achievement, the size (2 to 20 residues) and nature of ligands identified would not seem to be immediately applicable as drug candidates.
Combinatorial chemistry, introduced for polypeptide and oligonucleotide libraries, has undergone a rapid development and acceptance. It is widely recognized that this approach, when applied to generating non-peptide small molecule diversity, has provided a new paradigm for drug discovery. Perhaps as a consequence of the extension of the concept from peptide and oligonucleotide synthesis, the majority of applications have relied on solid-phase synthesis and methodological advances continue to extend common synthetic transformations to polymer-supported versions (Thompson et al. J. A. Chem. Rev. 1996, 96, 555; Frxc3xcchtel et al. Angew. Chem., Int. Ed. Engl. 1996, 35, 17; Hermkens et al. Tetrahedron 1996, 52, 4527)
A less well accepted complement to adapting solution-phase chemistry to polymer-supported combinatorial synthesis is the development of protocols for solution-phase combinatorial synthesis (Han et al. Proc. Natl. Acad. Sci. U.S.A. 1995, 92, 6419) Preceding the disclosure of our own efforts on the development of a multi-step solution-phase parallel synthesis of chemical libraries (Cheng et al. J. Am. Chem. Soc. 1996, 118, 2567; Boger et al. J. Am. Chem. Soc. 1996, 118, 2109; Cheng et al. Bioorg. Med. Chem. 1996, 4, 727), the single-step solution-phase synthesis of combinatorial libraries was detailed by at least three groups as follows. Smith and coworkers (Smith et al. Bioorg. Med. Chem. Lett. 1994, 4, 2821), prepared a library of potentially 1600 amides by reacting 40 acid chlorides with 40 nucleophiles. The library was screened as 80 sample mixtures in a matrix format, allowing immediate deconvolution.
A similar sub-library format was used by Pirrung and Chen (Pirrung et al. J. Am. Chem. Soc. 1995, 117, 1240; Pirrung et al. Chem. Biol. 1995, 2, 621) who prepared a series of carbamate mixtures which were screened for acetylcholinesterase inhibitory activity. Prior to these efforts, Rebek""s group reported the single-step construction of large libraries presenting amino acid derivatives attached to rigid core templates with a reliance on amide or urea bond formation (Carell et al. Angew. Chem., Int. Ed. Engl. 1994, 33, 2059; Carell et al. Bioorg. Med. Chem. 1996, 4, 655; Dunayevskiy et al. Anal. Chem. 1995, 67, 2906; Carell et al. Chem. Biol. 1995, 2, 171). Because of the complexity of the combinatorial libraries resulting from this approach (approaching 100,000 members), an iterative selection strategy based on structural grouping of the building blocks was devised.
In addition to recent advances in this work, substantial progress towards using solution-phase multicomponent reactions for generating combinatorial mixtures has been disclosed. For example, both Ugi and Armstrong have reported Ugi four-component condensations including the incorporation of a modifiable isocyanide in combination with resin capture strategy, to provide useful solution-phase library preparations (Ugi et al. Endeavour 1994, 18, 115; Keating et al. J. Am. Chem. Soc. 1996, 118, 2574; Armstrong et al Acc. Chem. Res. 1996, 29, 123).
Our own efforts have focused on the development of a multistep, solution-phase strategy for the preparation of chemical libraries which relies upon the simple removal of excess reactants and reagents by liquid-liquid or liquid-solid extraction procedures. The application of water-soluble coupling reagents in solution-phase peptide synthesis was introduced by Sheehan et al. J. Org. Chem. 1956, 21, 439.
The approach has been shown to dependably deliver pure, individual compounds in multi-milligram quantities, and chemical libraries of  greater than 1000 individual members have been assembled (Cheng et al. J. Am. Chem. Soc. 1996, 118, 2567; Boger et al. J. Am. Chem. Soc. 1996, 118, 2109; Cheng et al. Bioorg. Med. Chem. 1996, 4, 727; Tarby et al. In Molecular Diversity and Combinatorial Chemistry: Libraries and Drug Discovery Chaiken, I. M., Janda, K. D., Eds.; ACS: Washington, 1996; 81). Notably, it avoids the disadvantages of solid-supported synthesis including its restrictive scale, the required functionalized substrates and solid supports, compatible spacer linkers, and the requirements for othogonal attachment/detachment chemistries typically with the release of spectator functional groups. It does not require specialized protocols for monitoring the individual steps of multistep syntheses including orthogonal capping strategies for blocking unreacted substrate and does provide the purification of sequence intermediates. This latter disadvantage of solid-supported synthesis necessarily produces the released product of a multistep sequence in an impure state or requires that each reaction on each substrate proceed with an unusually high efficiency.
Ligand-induced receptor and protein dimerization or oligomerization has emerged as a general mechanism for signal transductionl and members of the important receptor superfamilies are activated by such a process. These include protein tyrosine kinase receptors (homo- or heterodimerization), class I cytokine receptors (homo- or heterodimerization), serine/threonine kinase receptors (hetero-oligomerization), and members of the TNF-receptor family (trimerization), FIG. 20. Within the cytokine receptor superfamily, the most extensively studied examples are the human growth hormone (hGHr), prolactin (PRLr) and erythropoietin receptors (EPOr), which form homodimers upon binding their ligands. Similarly, intracellular signal transduction often proceeds by protein-protein homo- or heterodimerization and important examples include activators of transcription (e.g., Myc-Max dimerization, STAT homo- and heterodimers).
Important therapeutic applications may emerge from either the development of agonists or antagonists of such receptor or protein dimerization and representative examples are provided in FIG. 21 for the cytokine receptor superfamily. Our interest in combinatorial chemistry rested on its potential. ability to provide candidate leads for promoting receptor activation by dimerization which to our knowledge had not emerged from screening natural products. This interest in studying receptor activation via dimerization and the potential of utilizing a single approach for the discovery of antagonists and their conversion to agonists was one important element underlying our pursuit of solution-phase combinatorial chemistry at a time when solid-phase techniques were considered most valuable.
What is needed are small molecule libraries of C2-symmetric and unsymmetric chemical compounds for use in protein and receptor homodimerization and heterodimerization studies as described above. Furthermore, what is needed is an economical method for the rapid and multi-milligram preparation of said targeted C2-symmetric and unsymmetric chemical libraries.
One aspect of the invention is directed to a convergent process for synthesizing a symmetric combinatorial library. In the first step of the process, a template and n addition reagents, wherein n is greater than 3, are provided. The template is of a type which includes a linkage site and at least two addition sites. The linkage site is employable for dimerizing the template. Each of the addition sites is employable for conjugating the addition reagents onto the template. A combinatorial sublibrary is then constructed by conjugating each of the addition sites of the template with a subset of the n addition reagents in a combinatorial fashion. The combinatorial sublibrary constructed in the prior step is then dimerized by cross linking the linkage site for constructing the symmetric combinatorial library. In a preferred mode of the invention, both the conjugation and cross linking steps occur in solution phase. In an alternative preferred mode, a cross linking agent is employed for cross linking the linkage sites. Symmetric combinatorial libraries constructed by the above convergent process are further aspects of the invention.
Another aspect of the invention is directed to a convergent process for synthesizing an asymmetric combinatorial library. In the first step of the process, a first template, a second template, and n addition reagents, wherein n is greater than 3, are provided. The first template has a first linkage site and at least two addition sites. The second template has a second linkage site and at least two addition sites. The first and second linkage sites are employable for linking the first template to the second template. Each of the addition sites are employable for conjugating the addition reagents onto the template. A first combinatorial sublibrary is then constructed by conjugating each of the addition sites of the first template with a first subset of the n addition reagents in a combinatorial fashion. A second combinatorial sublibrary is then constructed by conjugating each of the addition sites of the second template with a second subset of the n addition reagents in a combinatorial fashion. The first and second combinatorial sublibraries constructed in the above two conjugation steps are then dimerized by cross linking the first linkage site on the first template of the first combinatorial sublibrary with the second linkage site on the second template of the second combinatorial sublibrary, so as to construct the asymmetric combinatorial library. In a preferred mode of the invention, both the conjugation and cross linking steps occur in solution phase. In an alternative preferred mode, a cross linking agent is employed for cross linking the linkage sites. Asymmetric combinatorial libraries constructed by the above convergent process are further aspects of the invention.
Another aspect of the invention is directed to a convergent process for synthesizing a double dimerized combinatorial library. In the first step of the process, a first template, a second template, a third template, a fourth template and n addition reagents, wherein n is greater than 3, are provided. The first template has a first linkage site and at least two addition sites. The second template has a second linkage site and at least two addition sites. The first and second linkage sites are employable for linking the first template to the second template. Each of the addition sites are employable for conjugating the addition reagents onto the template. A first combinatorial sublibrary is then constructed by conjugating each of the addition sites of the first template with a first subset of the n addition reagents in a combinatorial fashion. A second combinatorial sublibrary is then constructed by conjugating each of the addition sites of the second template with a second subset of the n addition reagents in a combinatorial fashion. A third combinatorial sublibrary is then constructed by conjugating each of the addition sites of the third template with a third subset of the n addition reagents in a combinatorial fashion. A fourth combinatorial sublibrary is then constructed by conjugating each of the addition sites of the fourth template with a fourth subset of the n addition reagents in a combinatorial fashion. The first and second combinatorial sublibraries are then dimerized by cross linking the first linkage site on the first template of the first combinatorial sublibrary with the second linkage site on the second template of the second combinatorial sublibrary. Dimerization is achieved using a first cross linking agent having a first supplemental linkage site for cross linking the first cross linking agent. The first supplemental linkage site is of a type which is unreactive with the linkage site of the first and second templates. The dimerization achieves the construction of a first intermediate combinatorial library. The third and fourth combinatorial sublibraries are then dimerized by cross linking the third linkage site on the third template of the third combinatorial sublibrary constructed with the fourth linkage site on the fourth template of the fourth combinatorial sublibrary. Dimerization is achieved using a second cross linking agent having a second supplemental linkage site for cross linking the second cross linking agent. The second supplemental linkage site is of a type which is unreactive with the linkage site of the third and fourth templates. The dimerization achieves the construction of a second intermediate combinatorial library. The double dimerized combinatorial library is then produced by linking the first supplemental linkage of the first intermediate combinatorial library with the second supplemental linkage of the second intermediate combinatorial library. In a preferred mode of the invention, both the conjugation and cross linking steps occur in solution phase. In an alternative preferred mode, a cross linking agent is employed for cross linking the linkage sites. Double dimerized combinatorial libraries constructed by the above convergent process are further aspects of the invention.
Another aspect of the invention is directed to a process for converting a first combinatorial library having an antagonist activity with respect to a receptor to a second combinatorial library having an agonist activity with respect to the same receptor. The receptor is of a type which is activated by dimerization. In the first step of the process, the first combinatorial library is provided. The first combinatorial library has n elements. At least one of the n elements has the antagonist activity with respect to the receptor. Each of the n elements has a linkage site for dimerization. The first combinatorial library is then dimerized by cross linking the linkage sites of the n elements with one another, including elements having the antagonist activity, for producing the second combinatorial library. Upon dimerization, the elements having antagonist activity in the first combinatorial library are converted to elements having agonist activity in the second combinatorial library. In a preferred mode of the invention, the cross linking step occurs in solution phase. In an alternative preferred mode, a cross linking agent is employed for cross linking the linkage sites. Agonist combinatorial libraries having an agonist activity with respect to a receptor, the receptor being of a type which is activated by dimerization, are further aspects of the invention.
Another aspect of the invention is directed to a deletion method for deconvoluting combinatorial libraries.